Enzymatic oxidative deamination process

ABSTRACT

The present invention concerns an enzymatic oxidative deamination process of a dipeptide monomer to prepare an intermediate useful to prepare compounds having endopeptidase and angiotensin converting enzyme inhibition activity.

This application is a divisional of U.S. Ser. No. 09/386,588 filed Aug.31, 1999, issued as U.S. Pat. No. 6,261,810, which claims priority fromprovisional application U.S. Serial No. 60/098,956 filed Sep. 3, 1998,both of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

The present invention concerns an enzymatic oxidative deaminationprocess to prepare an intermediate useful to prepare compounds havingendopeptidase and angiotensin converting enzyme inhibition activity.

BACKGROUND OF THE INVENTION

Robl in U.S. Pat. No. 5,508,272 discloses compounds of the formula

wherein A is

as possessing neutral endopeptidase and angiotensin converting enzymeinhibition activity. Among these compounds is[4S-[4α(R*),7α,10aβ]]-octahydro-4-[(2-mercapto-1-oxo-3-phenylpropyl)amino]-5-oxo-7H-pyrido[2,1-b][1,3]thiazepine-7-carboxylicacid which is currently undergoing clinical evaluation. This compound isreported herein as (1).

Robl discloses that the amino lactam portion of (1), i.e., theintermediate

can be prepared by coupling (S)-2-amino-6,6-dimethoxyhexanoic acidmethyl ester with the N-protected amino acid

wherein P₁ is an amino protecting group and P₂ is a sulfur protectinggroup to give the dipeptide of the formula

Removal of the P₂ protecting group, followed by acid catalyzedcyclization, and removal of the P₁ protecting group gives[4S-(4α,7α,10aβ)]-octahydro-4-amino-5-oxo-7H-pyrido[2,1-b][1,3]thiazephine-7-carboxdylicacid, methyl ester.

Robl discloses preparing (S)-2-amino-6,6-dialkoxyhexanoic acid, alkylester, such as (S)-2-amino-6,6-dimethoxyhexanoic acid, methyl ester, byconverting N-protected L-ε-hydroxynorleucine to its methyl ester,oxidizing to a corresponding aldehyde, such as of the formula

then reacting with trimethyl orthoformate in the presence of a strongacid catalyst, and removing the P₃ protecting group.

SUMMARY OF THE INVENTION

The present invention provides an enzymatic process for the preparationof the above-described intermediate, i.e., the protected amino lactamportion of (1).

More specifically, the present invention is directed to a process forpreparing an amino lactam compound of the formula I

comprising contacting a dipeptide monomer of the formula II

wherein P₁ is an amino protecting group, and R¹ is H, alkyl or of theformula

wherein R² is alkyl,

with an aminotransferase enzyme in the presence of α-ketoglutarate underconditions suitable for formation of the compound of formula I. Theimmediately preceding process will be referred to herein as the“oxidative deamination” process.

In an alternate embodiment, the oxidative deamination process isperformed in the presence of a glutamate oxidase enzyme which functionsto recycle glutamate formed during the process back toalpha-ketoglutarate.

The present invention is also directed to a process for preparing adipeptide monomer starting compound of the formula III

comprising contacting a dipeptide dimer compound of the formula IV

wherein P₁ is as defined above,

with a reducing agent under conditions suitable for formation of thecompound of formula III. The immediately preceding process for preparingthe dipeptide monomer will be referred to herein as the “reduction”process.

The present invention also concerns an engineered host cell containingrecombinant nucleic acid capable of expressing an aminotransferaseenzyme. In addition, the invention concerns a novel aminotransferasefrom Spingomonas sp. and nucleic acid encoding same.

The present invention also concerns the novel compounds of formulas II,III and IV.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1: DNA sequence (SEQ.ID.NO:1) encoding the preferredaminotransferase of the invention from Sphingomonas paucimobillis ATCC202027 and the amino acid sequence (SEQ.ID.NO.:2) of the preferredaminotransferase of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the above formulas the amino protecting group, P₁, is a group such asbenzyloxycarbonyl, phenoxyacetyl, phenyl acetyl, phenylmethoxycarbonyl,t-butyloxycarbonyl or the like; or a group which together with theN-atom forms a protecting group such as phthalimido. The most preferredP₁ group is phenylmethoxycarbonyl which is sometimes referred to hereinas “Cbz.”

After formation of the compound of formula I, said compound can bedeprotected by techniques well known in the art to afford the abovedescribed intermediate, i.e., the amino lactam portion of (1). Forexample, when P₁ is phenylmethoxycarbonyl, treatment withiodotrimethylsilane or t-butoxycarbonyl treatment with a strong acidsuch as hydrochloric acid can be employed; when P₁ is phenoxyacetyl orphenylacetyl, the deprotection can be done enzymatically usingPenicillin G acylase or Penicillin v acylase, and the like.

Although it is possible to perform the reduction process, isolate thecompound of the formula III, and subsequently perform the oxidativedeamination process, it is preferred to perform both steps in the samereactive medium wherein all components necessary to produce the compoundof the formula I starting with compound of the formula IV are present.In such a situation, the reduction process and oxidative deaminationprocess can be viewed as part of a single process.

The term “alkyl” or “alk” as used herein alone or as a part of anothergroup, denotes such optionally substituted, but preferablyunsubstituted, straight and branched chain saturated hydrocarbon groups,preferably having 1 to 10 carbons in the normal chain. Exemplaryunsubsituted such groups include methyl, ethyl, propyl, isopropyl,n-butyl, iso-butyl, t-butyl, pentyl, hexyl, isohexyl, heptyl,4,4-dimethyl, pentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl and thelike. The alkyl groups may be substituted by appropriate substituentsproviding compounds suitable for use in the present invention. Exemplarysubstituents of the alkyl group include one or more, preferably three orfewer, chloro groups, bromo groups, or iodo groups. The alkyl groups ofR¹ and R² are preferably unsubstituted lower alkyl and most preferablymethyl.

The term “lower alkyl” as used herein denotes alkyl groups having 1 to 3carbon atoms.

The aminotransferase enzyme employed in the present invention may be anyaminotransferase enzyme capable of catalyzing the conversion of CompoundII into Compound I. Optionally, a glutamate oxidase enzyme may beemployed which can be any glutamate oxidase enzyme capable of convertingglutamate to alpha-keto glutarate. Enzymatic or microbial materials asthe source of either of the enzymes (i.e., the aminotransferase and/orglutamate oxidase) may be employed in the free state or immobilized on asupport such as by physical absorption or entrapment.

Suitable enzymes, regardless of origin or purity, are those enzymesreferred to as aminotransferase enzyme and glutamate oxidase enzymes.The enzymes employed may, for example, be an enzyme isolated from amicroorganism such as by homogenizing cell suspensions, followed bydisintegration, centrifugation, DEAE-cellulose chromatography, ammoniumsulfate fractionation, chromatography using gel filtration media such asSephacryl(crosslinked co-polymer of allyl dextran and N,N′-methylenebisacrylamide) chromatography, and ion exchange chromatography such asMono-Q (anion exchanger which binds negatively charged biomoleculesthrough quaternary amine groups) chromatography.

Alternatively, the oxidative deamination process may use intact cells orcell extracts as a source of the enzyme(s). With respect to the use ofmicroorganisms, the process of the present invention may be carried outusing any suitable microbial materials capable of catalyzing the desiredreaction. For example, the cells may be used in the form of intact wetcells or dried cells such as lyophilized, spray-dried or heat-driedcells, or in the form of treated cell material such as ruptured cells orcell extracts. Suitable microorganisms include genera from bacteria,yeasts and fungi that contain either endogenous or recombinant nucleicacid encoding either or both of the aminotransferase and glutamateoxidase enzymes. Examples of suitable organisms include: Bacillus sp.such as B. subtilis; Sporosarcina sp.; Escherichia coli, Pichia sp. suchas Pichia pastoris; Thermoactmomyces sp. such as T. intermedius;Pseudomonas sp.; Spingomonas sp. such as Spingomonas paucimobilis;Streptomyces sp. such as Streptomyces noursei; Candida sp.;Saccharomyces sp.; Cephalosporium sp.; Fusarium sp.; Penicillium sp.;and the like.

The use of genetically engineered organisms is specificallycontemplated. The host cell may be any cell, e.g., the ones mentioned inthe immediately preceding paragraph such as Escherichia coli, Pichiapastoris, Streptomyces Sp., and the like, modified to contain a gene orgenes for expressing one or more aminotransferases and/or glutamateoxidases capable of catalysis as described herein.

It is particularly preferred to employ microorganisms of the Pseudomonasand Sphingomonas genera, particularly the species Sphingomonaspaucimobilis, especially the strain Sphingomonas paucimobilis ATCC202027 (formerly known as Pseudomonas SC 16133) for the source of theaminotransferase. It is particularly preferred to employ microorganismsof the genus Streptomyces, as the source of the glutamate oxidase. Theterm “ATCC” as used herein refers to the accession number of theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Sphingomonas paucimobilis ATCC 202027 was deposited withthe ATCC on Sep. 9, 1997, under the provisions of the Budapest Treaty.

A commercial source of a glutamate oxidase useful in the oxidativedeamination process is Sigma Chemical Co., St. Louis, Mo., productnumber GO400.

The oxidative deamination process of the present invention may becarried out subsequent to the fermentation of the microorganism(s)employed (two-stage fermentation and conversion), or concurrentlytherewith, that is, in the latter case, by in situ fermentation andreduction (single-stage fermentation and reduction). In the single-stageprocess, the microorganisms may be grown in an appropriate medium untilsufficient growth of the microorganisms is attained. A compound offormula II may then be added to the microbial cultures and the oxidativedeamination continued with the fermentation, preferably until completeconversion to the compound of formula I is obtained.

In the two-stage process, the microorganisms may, in the first stage, begrown in an appropriate medium for fermentation until exhibiting thedesired enzymatic activity. Subsequently, the cells may be harvested bycentrifugation and microbial cell suspensions prepared by suspendingharvested cells in an appropriate buffered solution. Buffers such asTris-HCl, phosphates, sodium acetate and the like may be used. Water mayalso be used to prepare suspensions of microbial cells. In the secondstage, the compound of formula II may be mixed with the microbial cellsuspensions, and the enzymatic conversion of the compound catalyzed bythe microbial cell suspension. The reaction is preferably conducteduntil all or nearly all of the compound of formula II is converted tothe compound of formula I.

Growth of the microorganisms may be achieved by one of ordinary skill inthe art by the use of an appropriate medium. Appropriate media forgrowing microorganisms include those which provide nutrients necessaryfor the growth of the microbial cells. A typical medium for growthincludes necessary carbon sources, nitrogen sources, and trace elements.Inducers may also be added. The term “inducer”, as used herein, includesany compound enhancing formation of the desired enzymatic activitywithin the microbial cell, such as those compounds containing L-lysineand L-lysine analogs. Formula I compounds may be added as inducersduring growth of the microorganism.

Carbon sources may include sugars such as maltose, lactose, glucose,fructose, glycerol, sorbitol, sucrose, starch, mannitol, propyleneglycol, and the like; organic acids such as sodium acetate, sodiumcitrate, and the like; amino acids such as sodium glutamate, and thelike; and alcohols such as methanol, ethanol, propanol, and the like.

Nitrogen sources may include N-Z amine A, corn steep liquor, soy beanmeal, beef extracts, yeast extracts, yeastamin, molasses, baker's yeast,tryptone, nutrisoy, peptone, sodium nitrate, ammonium sulfate, and thelike.

Trace elements may include phosphates and magnesium, manganese, calcium,cobalt, nickel, iron, sodium and potassium salts.

The medium employed may include more than one carbon or nitrogen sourceor other nutrient.

A preferred medium for Streptomyces sp. is an aqueous medium having thefollowing components (in weight %):

Component Nutrisoy 3% Maltrin M 180 1%

A preferred medium for Sphingomonas paucimobilis ATCC 202027 is anaqueous medium having the following components (in weight %):

Component Peptone 1.5% Yeast Extract 1.0% KH₂PO₄ 0.2% K₂HPO₄ 0.2% MgSO₄0.01% NaCl 0.2%

A preferred medium for E. coli is an aqueous medium having the followingcomponents (in weight %):

Component NZ Amine A 1% Yeastamin 2% Glycerol 2% Na₂HPO₄ 0.6% K₂HPO₄0.3% (NH₄)₂SO₄ 0.125 Propylene glycol 0.05% MgSO₄ · 7H₂O 0.0246%Kanamycin 0.005% pH 7.0-7.2

The pH of the medium is preferably adjusted to about 6 to 8, dependingupon the particular medium, sterilized, e.g., at a temperature of 121°C. for 30 minutes, and then adjusted to a desirable pH aftersterilization.

When growing microorganisms, the oxidative deamination process of theinvention is preferably carried out under aerobic conditions. Theagitation and aeration of the reaction mixture affects the amount ofoxygen available during the process which may be conducted, for example,in shake-flask cultures or fermentor tanks during growth ofmicroorganisms in a single-stage or two-stage process. The agitationrange from 50 to 500 RPM is preferable, with 50 to 100 RPM being mostpreferred. Aeration of about 0.1 to 10 volumes of air per volume ofmedia per minute (i.e., 0.1 to 10 v/vt) is preferred, with aeration ofabout 5 volumes of air per volume of media per minute (i.e., 5 v/vt)being most preferred.

If the oxidative deamination process is performed in a second stageafter growth of the microorganisms, oxygen is not known to be requiredand may be detrimental.

The oxidative deamination process of the present invention is carriedout using alpha-ketoglutarate and, optionally, a glutamate oxidaseenzyme. The glutamate oxidase catalyzes the reaction of glutamate toform alpha-ketoglutarate and thereby providing a means of recycling thealpha-ketoglutarate. The alpha-ketoglutarate functions as an acceptor ofthe amino group of the compound of formula II.

In the oxidative deamination process the compound of the formula II isconverted to the compound of the formula I via two intermediates whichspontaneously convert to form Compound I, which is illustrated by thefollowing reaction scheme:

In the oxidative deamination process, the compound of the formula IIwith a free sulfhydryl, S-alkyl, or S-acetyl can be employed. In case ofthe S-alkylated compound of the formula II, the alkyl group is firstremoved either chemically or enzymatically and converted to a compoundof the formula II with free sulfhydryl and is then used in the enzymaticprocess. In case of the S-acetyl compound of the formula II, the basicreaction conditions causes hydrolysis of the acetyl group to generatethe desired compound of the formula II with a free sulfhydryl group.

It is preferred to employ an aqueous liquid as the reaction medium,although an organic liquid, or a miscible or immiscible (biphasic)organiclaqueous liquid mixture may also be employed.

It is preferred to employ 0.1 to 25 weight % of the compound of formulaII starting material based on the combined weight of the compound andreaction medium. The amount of enzyme or microorganism employed relativeto the starting material is selected to allow catalysis of the oxidativedeamination process.

The products of the oxidative deamination process of the presentinvention may be isolated and purified, if desired, by knownmethodologies such as by extraction distillation, crystallization,column chromatography, and the like.

A preferred method for separating the desired compound of formula I fromthe remaining compounds of the reaction medium is concentration byremoval of water, then addition of methanol to crystallize out the aminoacid.

Preferred aminotransferases useful herein are selected from those aminotransferases known as Epsilon aminotransferases andL-α-aminotransferases.

Specific examples include aspartate aminotransferases, glutamateaminotransferases, pyrurate aminotransferases, ornithineaminotransferases, branch-chain aminotransferases, and the like. Themost preferred aminotransferase has the sequence shown in FIG. 1(SEQ.ID.NO.:2). Other preferred aminotransferases have at least 80%identity to SEQ.ID.No.:2, more preferably at least 90% identity toSEQ.ID.NO.:2. Glutamate oxidase is commercially available fromStreptomyces sp. (Sigma Chemicals, Product #G0400).

The present invention also contemplates use of two or moreaminotransferases and/or two or more glutamate oxidases, particularlywhen using whole cells or crude extracts. The amino transferases andglutamate oxidases, useful in the present invention are typically from avariety of plant, animal, and microbial origins. Alternatively, theenzymes useful in the present invention may be obtained by syntheticmeans, i.e., chemical synthesis of the polypeptide from its componentamino acids, by methods known to those of ordinary skill in the art. Forexample, the solid phase procedure described in Houghton et al., Proc.Natl. Acad. Sci. 82, 5131-5135 (1985) may be employed. The enzymes maybe obtained by production in prokaryotic or eukaryotic host cellsexpressing a DNA sequence coding for the desired enzyme (endogenous orrecombinant), or by in vitro translation of the mRNA encoded by a DNAsequence coding for all or part of the desired enzyme. Techniques forthe production of polypeptides by these means are known in the art, andare described herein.

Conditions for the enzymatic oxidative deamination process can varywidely depending on the type and form of enzyme employed.

The oxidative deamination process of the present invention is performedunder conditions suitable for forming the desired compound of formula I.The pH of the medium is preferably maintained between about 4 and 12,more preferably between about 6 and 8, during the growth ofmicroorganisms. During the process, whether performed with enzymes ormicroorganisms, the pH is maintained between about 6 and 10, preferablybetween about 7 and 9.

Temperature is a measure of the heat energy available for the oxidativedeamination process, and should be maintained to ensure that there issufficient energy available for this process. A suitable temperaturerange for the process of the invention is from about 15° C. to about 60°C. A preferred temperature range is from about 25° to about 40° C. Ifthe process involves active fermentation, a suitable temperature rangeis about 35° C. to about 45° C., preferably about 40° C. A typicalreaction time is about 4 to about 48 hours, preferably about 4 to 8hours, measured from the time of initially treating the compound offormula II with a microorganism(s) or enzyme(s) as described herein. Itis preferred that complete or substantially complete conversion of thecompound of formula II takes place.

Pressure is not known to be critical to practice of the invention andfor convenience about atmospheric pressure is typically employed. Abroad pH range for the process is about 6 to about 10, preferably about7 to about 9, regardless of whether active fermentation is employed.

The oxidation deamination process of the present invention results inhigh yield of the compound of Formula I and Formula III. A typical yieldis greater than about 70%, preferably greater than about 80%, morepreferably greater than about 90%, and most preferably about 95%.

It is preferable to actively terminate the oxidative deaminationprocess. The reaction can be terminated by adding an effective amount ofacid to the reaction mixture. Preferred acids are strong acids such astrichloroacetic acid (TCA), methane sulfonic acid, phosphoric acid,acetic acid, hydrochloric acid, sulfuric acid, and the like. Aneffective amount of acid is sufficient to lower pH to about 2; e.g.,about 5% to 20% acid, preferably about 10% acid.

To prepare the starting dipeptide dimer of formula IV in the reductionprocess, L-homocystine is reacted with an appropriate protecting grouphalide (e.g., CBZ-Cl) to form a protected L-homocystine of the formula

For CBZ protected L-homocystine, the following scheme is illustrated:

The protected L-homocystine is then reacted with and dicyclohexylcarbodimide (DCC) to form N-[(Phenylmethoxy)carbonyl]-L-homocysteine,(1→1′)-disulfide. The compound so formed is then reacted withN-MBOC-L-Lysine to form the dipeptide dimer of Formula IV. The compoundof Formula IV is then contacted with a reducing agent under conditionssuitable for formation of the compound of formiua III. Reducing agentsinclude dithiothreitol (DTT), 2-mercaptoethanol, tributylphosphine, andthe like. For CBZ protected compounds, the following scheme isillustrated:

Suitable conditions for the reduction process include a temperature ofabout 25° C. to about 40° C., a reaction time of about 5 minutes toabout 1 hour, and a pH of about 7 to about 9. Suitable solvents for thereduction process include water;water containing 10% solvent, such asethanol, methanol, or acetonitrile; and the like.

As described by Robl in U.S. Pat. No. 5,508,272,(S)-2-amino-6,6-dimethoxyhexanoic acid, methyl ester of formula X can becoupled with the N-protected amino acid of the formula V

to give the dipeptide of the Formula VI

wherein P₁ is an amino protecting group such as benzyloxycarbonyl ort-butyloxycarbonyl, phenoxyacetyl, phenylacetyl, or a group whichtogether with the N-atom forms a protecting group such as phthalimidoand P₂ is a mercapto protecting group such as acetyl or benzoyl. Thiscoupling reaction is preferably performed in the presence of a couplingreagent such as benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate, ethyl-3-(3-dimethyl-amino)propyl carbodiimide,dicyclohexylcarbodiimide, or methanesulfonyloxybenzotriazole.

The P₂ protecting group is selectively removed from the dipeptide offormula VI such as by treatment with sodium methoxide in methanol or bytreatment with p-toluenesulfonic acid in methanol. The resultingmercaptan compound is then subjected to an acid catalyzed cyclizationreaction preferably by treating with a strong acid such astrifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, or acommercially available polystyrene sulfonate polymer type ion exchangeresin such as Amberlyst15®. This cyclization reaction can be performedin a non-protic solvent such as methylene chloride or chloroform to givethe lactam of the formula VII

The lactam of formula VII is then treated to remove the P₁ N-protectinggroup and then reacted with the acylmercaptoalkanoyl sidechain of theformula VIII

wherein R₆ is methyl or phenyl giving the compound of the formula IX

This coupling reaction can be per formed in an organic solvent such asmethylene chloride and in the presence of a coupling reagent such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide,benzotriazol-1-yloxytris-(dimethylamino)phosphonium hexafluorophosphate,or carbonyldiimidazole. Alternatively, the acylmercaptoalkanoic acid offormula VIII can be converted to an activated form prior to couplingsuch as an acid chloride, mixed anhydride, symmetrical anhydride,activated ester, etc.

The P₁ N-protecting group can be removed from the lactam of formula VII,for example, by treatment with hydrazine monohydrate when P₁ togetherwith the N-atom forms a phthalimido group or by treatment withiodotrimethylsilane or palladium on carbon and hydrogen when P₁ isbenzyloxycarbonyl or by treatment with hydrochloric acid in dioxane orother strong acid when P₁ is t-butoxycarbonyl.

The acyl group R₆—C(O)— is removed and the methyl ester group isconverted to the carboxylic acid from the compound of formula IX to givethe desired final product of formula XI. For example, when R₆ is methyltreatment with methanolic sodium hydroxide followed by aqueous acidyield the desired compound of formula XI.

4S-[4α(R*),7α,10aβ]]-Octahydro-4-[(2-mercapto-1-oxo-3-phenylpropyl)amino]-5-oxo-7H-pyrido[2,1-b][1,3]thiazepine-7-carboxylicacid possesses angiotensin converting enzyme and neutral endopeptidaseinhibitory activity. This compound as well as its pharmaceuticallyacceptable salts are useful in treating cardiovascular diseases such ashypertension and congestive heart failure as noted in Robl U.S. Pat. No.5,508,272. This compound can be administered to a mammalian host such asman at from about 0.1 mg to about 100 mg per kg of body weight per day,preferably from about 0.5 mg to about 25 mg per kg of body weight perday. The compound is preferably administered orally but parenteralroutes and topical routes can also be employed. The daily dose can beadministered singly or can be divided into two to four dosesadministered throughout the day.

The present invention also concerns an engineered host cell comprising arecombinant nucleic acid sequence coding for aminotransferase derivedfrom Sphingomonas paucimobilis ATCC 202027 (preferably SEQ.ID.NO.:1).Preferably, the nucleic acid molecules are DNA molecules and the nucleicacid sequences are DNA sequences.

As used in the present application, the term “modified”, when referringto a nucleotide or polypeptide sequence, means a nucleotide orpolypeptide sequence which differs from the wild-type sequence found innature.

The recombinant host cell of the present invention can be anymicroorganism that is capable of producing recombinant aminotransferaseand is capable of being transformed or genetically engineered withaminotransferase from a different species to express catalyticallyactive aminotransferase. Examples of host cells of the inventioninclude, for example, Bacillus such as B. subtilis; Sporosarcina;Escherichia coli; Pichia such as Pichia pastoris; Thermoactmomyces suchas T. intermedius, Pseudomonas.; Sphingomonas such as Sphingomonaspaucimobilis; Streptomyces such as Streptomyces noursei; Candida;Saccharomyces, Cephalosporium.; Fusarium ; Penicillium ; and the like. Apreferred host cell of the invention is Escherichia coli.

All DNA sequences are represented herein by formulas whose left to rightorientation is in the conventional direction of 5′ to 3′. Nucleotidebase abbreviations used herein are conventional in the art, i.e., T isthymine, A is adenine, C is cytosine, and G is guanine; also, X isA,T,C, or G, Pu is purine (i.e., G or A), and Py is pyrimidine (i.e., Tor G). Further preferred as the DNA for the recombinant aminotransferaseis a DNA sequence having all or part of the nucleotide sequencesubstantially as shown in FIG. 1; or a DNA sequence complementary tothis DNA sequence; or a DNA sequence which hybridizes to a DNA sequencecomplementary to one of these DNA sequences. Preferably, the DNAsequence hybridizes under stringent conditions. Stringent hybridizationconditions select for DNA sequences of greater than 80% identity,preferably greater than 85% or, more preferably, greater than 90%identity. Screening DNA under stringent conditions may be carried outaccording to the method described in Nature, 313: 402-404 (1985). TheDNA sequences capable of hybridizing under stringent conditions with theDNA disclosed in the present application may be, for example, allelicvarlants of the disclosed DNA sequences, or may be derived from otherbacterial, fungal or yeast sources. General techniques of nucleic acidhybridization are disclosed by Sambrook et.al., In: Molecular Cloning, aLaboratory Manual, 2d edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984), and by Haymes et al., In: Nucleic AcidHybridization, a Practical Approach, IRL Press, Washington, D.C. (1985),which references are incorporated herein by reference. Stringenthybridization conditions are described in Hames et al. at pages 119-121as the following: Incubation in 4×SET buffer, 10×Denhardt's solution,0.1% SDS, 0.1% sodium pyrophosphate, 50 μg/ml denatured salmon sperm DNAat 68° C. for 16-24 hours; followed by washing three times for 20minutes in 3×SET, 0.1% SDS, 0.1% sodium pyrophosphate at 68° C., washingtwice for 20 minutes in 1×SET, 0.1% SDS, 0.1% sodium pyrophosphate at68° C., washing once for 20 minutes in 0.1×SET, 0.1% SDS, 0.1% sodiumpyrophosphate at 68° C., and washing once for 20 minutes in 4×SET atroom temperature. In the case of a nucleotide sequence (e.g., a DNAsequence) coding for part of the enzyme, it is required that thenucleotide sequence code for a fragment that is catalytically active,i.e. has enzymatic activity.

The preferred DNA encoding aminotransferase has the sequence of SEQ.I.D.No. 1, its complement, or a DNA molecule capable of hybridyzing understringent conditions to a DNA having the sequence of SEQ. I.D. NO:1 orits complement. The preferred aminotransferase of the invention has theamino acid sequence of SEQ. I.D. NO:2 or an amino acid sequence havingat least 80% identity to SEQ.I.D. NO:2, more preferably at least 90%identity.

The variant amino acid or DNA sequences within the scope of theinvention are homologous to the sequences specifically disclosed herein.The degree of homology (percent identity) between a specificallydisclosed and a variant sequence may be determined, for example, bycomparing the two sequences using the GAP computer programs, version6.0, described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl Math 2:482,1981). Briefly, the GAP program defines similarityas the number of aligned symbols (i.e., nucleotides or amino acids)which are similar, divided by the total number of symbols in the shorterof the two sequences. The preferred default parameters for the GAPprogram include: (1) a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

The aminotransferase sequence(s) of the present invention used totransform the host cell(s) can be obtained using various methodswell-known to those of ordinary skill in the art. At least threealternative principal methods may be employed:

(i) the isolation of a double-stranded DNA sequence from genomic DNA orcomplementary DNA (cDNA) which contains the sequence;

(2) the chemical synthesis of the DNA sequence; and

(3) the synthesis of the DNA sequence by polymerase chain reaction(PCR).

In the first approach, a genomic or cDNA library can be screened inorder to identify a DNA sequence coding for all or part ofaminotransferase. For example, a Sphingomonas paucimobilis genomic DNAlibrary can be screened in order to identify the DNA sequence coding forall or part of aminotransferase. Various techniques can be used toscreen the genomic DNA or cDNA libraries.

For example, labeled single stranded DNA probe sequences duplicating asequence present in the target genomic DNA or cDNA coding for all orpart of aminotransferase can be employed in DNA/DNA hybridizationprocedures carried out on cloned copies of the genomic DNA or cDNA whichhave been denatured to single stranded form.

A genomic DNA or cDNA library can also be screened for a genomic DNA orcDNA coding for all or part of aminotransferase using immunoblottingtechniques.

In one typical screening method suitable for either immunoblotting orhybridization techniques, the genomic DNA library, which is usuallycontained in a vector, or cDNA library is first spread out on agarplates, and then the clones are transferred to filter membranes, forexample, nitrocellulose membranes. A DNA probe can then be hybridized oran antibody can then be bound to the clones to identify those clonescontaining the genomic DNA or cDNA coding for all or part ofaminotransferase.

In the second approach, the DNA sequences of the present inventioncoding for aminotransferase can be chemically synthesized. For example,the DNA sequence coding for aminotransferase can be synthesized as aseries of 100 base oligonucleotides that can be sequentially ligated(via appropriate terminal restriction sites or complementary terminalsequences) so as to form the correct linear sequence of nucleotides.

In the third approach, the DNA sequences of the present invention codingfor aminotransferase can be synthesized using PCR. Briefly, pairs ofsynthetic DNA oligonucleotides at least 15 bases in length (PCR primers)that hybridize to opposite strands of the target DNA sequence are usedto enzymatically amplify the intervening region of DNA on the targetsequence. Repeated cycles of heat denaturation of the template,annealing of the primers and extension of the 3′-termini of the annealedprimers with a DNA polymerase results in amplification of the segmentdefined by the 5′ ends of the PCR primers. See, White et al., TrendsGenet. 5, 185-189 (1989).

The DNA sequences useful in the present invention coding foraminotransferase can also be modified (i.e., mutated) to prepare variousmutations. Such mutations may be either degenerate, i.e., the mutationchanges the amino acid sequence encoded by the mutated codon, ornon-degenerate, i.e., the mutation does not change the amino acidsequence encoded by the mutated codon. These modified DNA sequences maybe prepared, for example, by mutating the aminotransferase DNA sequenceso that the mutation results in the deletion, substitution, insertion,inversion or addition of one or more amino acids in the encodedpolypeptide using various methods known in the art. For example, themethods of site-directed mutagenesis described in Morinaga et al.,Bio/Technol. 2, 636-639 (1984), Taylor et al., Nucl. Acids Res. 13,8749-8764 (1985) and Kunkel, Proc. Natl. Acad. Sci. USA 82, 482-492(1985) may be employed. In addition, kits for site-directed mutagenesismay be purchased from commercial vendors. For example, a kit forperforming site-directed mutagenesis may be purchased from AmershamCorp. (Arlington Heights, Ill.). In addition, disruption, deletion andtruncation methods as described in Sayers et al., Nucl. Acids Res. 16,791-802 (1988) may also be employed. Both degenerate and non-degeneratemutations may be advantageous in producing or using the polypeptides ofthe present invention. For example, these mutations may permit higherlevels of production, easier purification, or provide additionalrestriction endonuclease recognition sites. All such modified DNA andpolypeptide molecules are included within the scope of the invention.

The host cells of the invention can be conveniently transformed by useof expression vectors comprising a DNA sequence coding foraminotransferase. The expression vectors preferably contain all or partof one of the DNA sequences having the enzyme(s) nucleotide sequencesubstantially as shown in Takada, H., et al, J. Biochem., 109, 371-376(1991). Further preferred are expression vectors comprising one or moreregulatory DNA sequences operatively linked to the DNA sequence codingfor all or part of aminotransferase. As used in this context, the term“operatively linked” means that the regulatory DNA sequences are capableof directing the replication and/or the expression of the DNA sequencecoding for aminotransferase.

Expression vectors of utility in the present invention are often in theform of “plasmids”, which refer to circular double stranded DNA loopswhich, in their vector form, are not bound to the chromosome. However,the invention is intended to include such other forms of expressionvectors which serve equivalent functions and which become known in theart subsequently hereto.

Expression vectors useful in the present invention typically contain anorigin of replication, a promoter located in front (i.e., upstream of)the DNA sequence (preferably an inducible promoter such as tac orbacteriophage λpL) and followed by the DNA sequence coding for all orpart of aminotransferase. The DNA sequence coding for all or part ofaminotransferase is followed by transcription termination sequences andthe remaining vector. The expression vectors may also include other DNAsequences known in the art, for example, stability leader sequenceswhich provide for stability of the expression product, secretory leadersequences which provide for secretion of the expression product,sequences which allow expression of the structural gene to be modulated(e.g., by the presence or absence of nutrients or other inducers in thegrowth medium), marker sequences which are capable of providingphenotypic selection in transformed host cells, stability elements suchas centromeres which provide mitotic stability to the plasmid, andsequences which provide sites for cleavage by restriction endonucleases.The characteristics of the actual expression vector used must becompatible with the host cell which is to be employed. For example, whencloning in a fungal cell system, the expression vector should containspromoters isolated from the genome of fungal cells (e.g., the trpCpromoter from Aspergillus nidulans, the AOX1 promoter from Pichiapastoris, and the GAP promoter from P. pastoris). Certain expressionvectors may contain an autonomously replicating sequence (ARS; e.g., ARSfrom Fusarium oxysporum, Saccharomyces cerevisiae, and the like) whichpromotes in vivo production of self-replicating plasmids in fungalhosts. It is preferred that the yeast expression vectors of theinvention do not have a yeast ARS sequence and thus will integrate intohost chromosomes upon plasmid entry of host cells. Such integration ispreferred because of enhanced genetic stability. An expression vector ascontemplated by the present invention is at least capable of directingthe replication in and integration in fungal cells, and preferably theexpression, of the PDH DNA sequence disclosed in Takada, H., et al, J.Biochem., 109, 371-376 (1991) in Pichia cells. Suitable promotersinclude, for example, the trpC promoter from Aspergillus nidulans, thepenicillin V amidase promoter from F. oxysporum, and the AOX promoterfrom P. pastoris. Suitable termination sequences include, for example,the trpC terminator from A. nidulans, the PVA terminator for F.oxysporum, and the AOX1 transcription termination sequence of P.pastoris. It is also preferred that the expression vector include asequence coding for a selectable marker. The selectable marker ispreferably antibiotic resistance. As selectable markers, G418 resistancecan be conveniently employed. All of these materials are known in theart and are commercially available.

Preferably, the host cell in which the DNA sequence encoding the enzymeis cloned and expressed is a prokaryotic such as E. coli. For example,E. coli K12 strain 294 (ATCC 31446), E. coli B, E. coli X1776 (ATCC31537), E. coli strain ST9 or E. coli JM 101 can be employed. Otherprokaryotes can also be used; for example, bacilli such as Bacillassubtilis and enterobacteriaceae such as Salmonella typhimurium orSerratia marcesans.

In general, where the host cell is a prokaryote, expression or cloningvectors containing replication and control sequences which are derivedfrom species compatible with the host cell are used. The vector may alsocarry marking sequences which are capable of providing phenotypicselection in transformed cells. For example, E. coli has commonly beentransformed using pBR322, a plasmid derived from an E. coli species(Bolivar et al., Gene 2:95 (1977)). pBR322 contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells.

For use in expression, the plasmid including the DNA to be expressedcontains a promoter. Those promoters most commonly used in recombinantDNA construction for use with prokaryotic hosts include the lactamase(penicillinase) and lactose promoter systems (Chang et al, Nature,275:615 (1978); Itakura et al. Science, 198:1056 (1977); Goeddel et al.,Nature 281:544 (1979)) and a tryptophan (trp) promoter system (Goeddelet al. Nucleic Acids Res., 8:4057 (1980); EPO Publ. No. 0036776). Whilethese are the most commonly used, other microbial promoters such as thetac promoter (Amann et al., Gene 25,167-178 (1983)) have beenconstructed and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally in operable relationship to genes in vectors (Siebenlist etal. Cell 20:269 (1980)).

Particularly preferred are the expression vectors designated pAL 781-LATdescribed herein below, which contain the DNA sequence coding foraminotransferase, or expression vectors with the identifyingcharacteristics of these plasmids.

Suitable expression vectors containing the desired coding and controlsequences may be constructed using standard recombinant DNA techniquesknown in the art, many of which are described in Sambrook et al.Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989).

The host cells of the invention preferably contain an expression vectorcomprising one or more regulatory DNA sequences capable of directing thereplication and/or the expression of and operatively linked to a DNAsequence coding for all or part of aminotransferase.

Expression vectors may be introduced into host cells by various methodsknown in the art. For example, transformation of host cells withexpression vectors can be carried out by the polyethylene glycolmediated protoplast transformation method. However, other methods forintroducing expression vectors into host cells, for example,electroporation, biolistic injection, or protoplast fusion, can also beemployed.

Once an expression vector has been introduced into an appropriate hostcell, the host cell may be cultured under conditions permittingexpression of large amounts of the desired polypeptide, in the preferredcase a polypeptide molecule comprising aminotransferase.

Host cells containing an expression vector which contains a DNA sequencecoding for all or part of aminotransferase may be identified by one ormore of the following five general approaches: (a) DNA-DNAhybridization; (b) the presence or absence of marker gene functions; (c)assessing the level of transcription as measured by the production ofmRNA transcripts in the host cell; (d) detection of the gene productimmunologically; and (e) enzyme assay, enzyme assay being the preferredmethod of identification.

In the first approach, the presence of a DNA sequence coding for all orpart of the desired enzyme can be detected by DNA-DNA or RNA-DNAhybridization using probes complementary to the DNA sequence.

In the second approach, the recombinant expression vector host systemcan be identified and selected based upon the presence or absence ofcertain marker gene functions (e.g., acetamide utilization, resistanceto antibiotics, resistance to fungicide, uracil prototrophy, etc.). Amarker gene can be placed in the same plasmid as the DNA sequence codingfor all or part of the enzyme under the regulation of the same or adifferent promoter used to regulate the enzyme coding sequence.Expression of the marker gene in response to induction or selectionindicates the presence of the entire recombinant expression vector whichcarries the DNA sequence coding for all or part of the desired enzyme.

In the third approach, the production of enzyme mRNA transcripts can beassessed by hybridization assays. For example, polyadenylated RNA can beisolated and analyzed by Northern blotting or nuclease protection assayusing a probe complementary to the RNA sequence. Alternatively, thetotal nucleic acids of the host cell may be extracted and assayed forhybridization to such probes.

In the fourth approach, the expression of all or part of the desiredenzyme can be assessed immunologically, for example, by Westernblotting.

In the fifth approach, expression of the enzyme can be measured byassaying for enzyme activity using known methods.

The DNA sequences of expression vectors, plasmids or DNA molecules ofthe present invention may be determined by various methods known in theart. For example, the dideoxy chain termination method as described inSanger et al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977), or theMaxam-Gilbert method as described in Proc. Natl. Acad. Sci. USA 74,560-564 (1977).

It should, of course, be understood that not all expression vectors andDNA regulatory sequences will function equally well to express the DNAsequences of the present invention. Neither will all host cells functionequally well with the same expression system. However, one of ordinaryskill in the art may make a selection among expression vectors, DNAregulatory sequences, and host cells using the guidance provided hereinwithout undue experimentation and without departing from the scope ofthe present invention.

The present invention further concerns a method for producingaminotransferase comprising culturing an engineered host cell containingrecombinant nucleic acid capable of expressing aminotransferase andcontaining an expression vector capable of expressing aminotransferase.Preferably the expression vector is pAL781-LAT.

All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature, J.Biol. Chem. 243, 3557-3559 (1969), abbreviations for amino acid residuesare as shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y TyrL-tyrosine G Gly L-glycine F Phe L-phenylalanine M Met L-methionine AAla L-alanine S Ser L-serine I Ile L-isoleucine L Leu L-leucine T ThrL-threonine V Val L-valine P Pro L-proline K Lys L-lysine H HisL-histidine Q Gln L-glutamine E Glu L-glutamic acid W Trp L-tryptophan RArg L-arginine D Asp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

All amino acid sequences are represented herein by formulas whose leftto right orientation is in the conventional direction of amino-terminusto carboxy-terminus.

It will be understood that allelic variations of the nucleic acid andamino acid sequences useful herein naturally exist, or may beintentionally introduced using methods known in the art. Thesevariations may be demonstrated by one or more amino acid differences inthe overall sequence, or by deletions, substitutions, insertions,inversions or additions of one or more amino acids in said sequence.Such amino acid substitutions may be made, for example, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphiphatic nature of the residues involved.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups or nonpolar headgroups having similar hydrophilicity values include the following:leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine,serine, threonine, phenylalanine, tyrosine. Other contemplatedvariations include salts and esters of the aforementioned polypeptides,as well as precursors of the aforementioned polypeptides, for example,precursors having N-terminal substituents such as methionine,N-formylmethionine and leader sequences. All such variations areincluded within the scope of the present invention.

The following examples illustrate the invention but should not beinterpreted as a limitation thereon.

All references cited herein are incorporated by reference in theirentirety.

EXAMPLE 1 Preparation ofN-[(Phenylmethoxy)carbonyl]-L-homocysteine,(1->1′)disulfide

To a 3-L, 5-necked flask fitted with a mechanical stirrer, thermocouple,pH electrode, pressure equalized addition funnel with CBZ-Cl, and asecond pressure equalized addition funnel with 1 N NaOH was charged100.0 g (0.373 mol) L-homocystine and 1 L of 1 N NaOH (solution pH was13.65). After cooling to 10° C., 117.0 mL (0.820 mol) CBZ-Cl was addeddropwise over 15 minutes, maintaining the temperature at 10 to 16° C.When the pH of the mixture dropped to 11, 500 mL of NaOH were addeddropwise. The pH after the addition of the 1 N NaOH was 7.89, requiring16 mL of 10 N NaOH to raise the pH of the mixture to 11.90. After thereaction was judged complete by in-process HPLC analysis (<1% change inarea counts of BMS-210474 disodium salt), 200 mL of MTBE were added andthe mixture was agitated for about 15 minutes. After the phases wereseparated, the aqueous phase was extracted with two, 200-mL portions ofMTBE. The aqueous solution was adjusted to pH 7.0 with 5.4 mL ofconcentrated HCl and the solution was sparged with nitrogen for 14hours. The pH of the solution was further adjusted to 6.11 with 60 mL of1 N HCl giving a very turbid suspension. The suspension was aged for 30minutes, during which time the pH rose to 6.28. To the suspension wasadded 65 mL of N HCl, giving a pH of 6.18. 50 mL of concentrated HClwere added to give a pH of 5.5. 200 mL of methanol were added and theslurry was heated to 43° C. to break up any crystal aggregates. The pHwas adjusted to 1.97 with 11 mL of concentrated HCl and the slurry wascooled to 20 to 25° C. After aging of the slurry for 2.5 hours, thecrystals were collected by vacuum filtration. The filter cake was washedwith three, 500-mL portions of water, followed by two, 250-mL portionsof heptanes to dewater the cake. The product was dried under reducedpressure at NMT 55° C., returning 191.0 g (0.356 mol, 95.4 M %) as awhite crystalline solid with a laboratory HPLC HI of 91.9

EXAMPLE 2 Preparation ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide

Procedure 1

To a 1-L, 3-necked flask equipped with a me chanical stirrer,thermocouple, and a nitrogen inlet and outlet is charged 30.00 g ofN-[(Phenylmethoxy)carbonyl]-L-homocysteine, (1->1′)-disulfide (55.91mmol, 1 eg.), 14.10 g N-hydroxysuccinimide (123.00 mmol, 2.2 eq.) and300 mL of DME. After the solution is cooled to −20 to −25° C., 29.99 gof DCC (145.37 mmol, 2.6 eq.) is added in one portion. The slurry isheld at −25 to −10° C. until the reaction is judged complete byin-process HPLC assay (21 hours, 0 relative area percentN-[(Phenylmethoxy)carbonyl]-L-homocysteine, (1->1′)-disulfide). Thesolids are filtered from the reaction and the filter cake is washed withtwo, 100-mL portions of DME. The solution is split into two, 250-mLportions.

To a 1-L, 5-necked flask equipped with a mechanical stirrer,thermocouple, pressure equalized addition funnel, and a nitrogen inletand outlet is charged 9.40 g sodium bicarbonate (111.89 mmol, 4 eq.) and200 mL of water. After dissolution of the bicarbonate, 17.21 g ofN-MBoc-L-lysine (69.89 mmol, 2.5 eq.) is added. The slurry is adjustedto pH 11.11 with −10 mL of 10 N NaOH to dissolve the lysine. Thesolution is cooled to 0° C. and the 250 mL solution of the active esteris added dropwise over 2.25 hours while maintaining the reactiontemperature at 0 to 5° C. After the reaction is judged complete byin-process HPLC analysis (˜0.5 hours), the solids are filtered from thesolution, and the cake is washed with two, 50-mL portions of DME. Thereaction solution is extracted with two, 100-mL portions of MTBE. Theproduct rich aqueous solution is diluted with 200 mL of MTBE andacidified to pH 1.64 with ˜16 mL of concentrated HCl. The phases aresplit and the organic phase is washed with 100 mL of water. The organicphase is concentrated on a rotary evaporator to a white, crystallinesolid of 28.08 g.

The solid is dissolved in 125 mL of DME and cooled to 0 to 5° C. 9 mL ofconcentrated sulfuric acid is added, maintaining the temperature below15° C. After the addition is complete, the solution is warmed to 20 to25° C. until the deprotection is judged complete by in-process HPLCanalysis. After the reaction is complete (˜0.75 hours), the solution isdiluted with 150 mL of water and concentrated to ˜one-half volume. Thesolution is diluted with 100 mL of water and extracted with two, 100 mLportions of MTBE.

The aqueous solution is added to ˜400 g of PVP resin (2.5 eq. resin pereq. sulfuric acid) and the resin slurry is diluted with 200 mL of waterand stirred with moderate agitation for 16 hours. The resin is removedby filtration over Hyflo andN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide is eluted with 500 mL portions of water. The fractionswith HPLC HI>90 are combined (fractions 8 through 23), concentrated on arotary evaporator, and lyophilized, to return 6.75 g (30.5 M %) ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide with a laboratory HPLC HI of 96.4

Procedure 2

Dicyclohexylcarbodimide (Aldrich, Lot KF 00929TZ; 4.82 g, 23.3 mmol) wasadded dropwise to a magnetically-stirred solution of L-CBZ-homocystine(6.22 g, 11.64 mmol) and N-hydroxysuccinimide (Aldrich, Lot JG 23818JG;2.7 g, 23.3 mmol) in 1,2-dimethoxy ethane (50 mL) while the mixture ismaintained at 0-5° C. The reaction mixture was stirred for 2 h at 0-5°C. and stored at 0° C. overnight. The reaction was followed by HPLC anddetermined to be complete after 18 h. The thick slurry was filtered andrinsed with 1,2-dimethoxy ethane (2×10 mL). The filtrate and thewashings were combined and used “as is” in the next step.

A mechanically-stirred suspension of N-M-Boc-lysine (Sigma, Lot 73H0151;5.74 g, 23.3 mmol) in water (55 mL) was cooled to 18° C. in an ice/waterbath and 1N NaOH (24 mL, 24 mmol) was added to it. The bis active ester(37102-170-17) was added as a solution in DME (100 mL, 11.64 mmol)dropwise over 30 minutes between 20-23° C. The pH was maintained between9 and 10 during the reaction by adding 1N NaOH. The reaction wasfollowed by HPLC and determined to be complete after 1.5 h. The reactionmixture was then extracted with MTBE (2×20 mL) and the neutral MTBEextract was set aside. The aqueous layer was diluted with MTBE (50 mL)and acidified with 1N HCl between 0-5° C. to pH 2. The organic layer wasseparated and the aqueous layer was extracted with MTBE (2×30 mL). TheMTBE extracts were combined, washed with water (2×40 mL) andconcentrated. The weight of the residue, obtained as a crystalline foam,was 11.14 g. The protected peptide dimer was used as is in the nextstep.

Concentrated sulfuric acid (1.9 mL) was added to a magnetically-stirredsolution of the protected peptide dimer (37102-171.22; 6 g, 6.04 mmol)in DME (20 mL) with cooling at 22° C. The reaction was followed by HPLCand found complete after 2 h. The reaction mixture was poured into 2:1.5water:DME (35 mL) and extracted with MTBE (2×15 mL). The aqueous phasewas adjusted to pH 4.25 using 10N NaOH. The HPLC of the aqueous phaseshowed 2 main peaks corresponding toN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide (76 HI) and the monolysine adduct (19 II). The aqueoussolution of N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide was then passed through a column of PVP resin (60 g)to remove sulfuric acid. The column was eluted with water and fractions2 to 10 (75 mL each) were pooled and concentrated. The concentratedaqueous solution ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide was then passed through a column of HP-20 resin (40g). Sodium sulfate was removed by eluting the column with water (200mL). The absence of sulfate in subsequent fractions was determined usinga BaCl₂ test. The column was then eluted with 1:1 MeOH:water (300 mL).Finally the column was eluted with MeOH and fractions 3 to 8 (100 mLeach) were pooled, concentrated and lyopholized to furnishN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide as a white powder (1.6 g). The overall yield beginningfrom Z-homocystine was 34%. Laboratory HPLC HI 91.

EXAMPLE 3 Aminotransferase Purification from Sphingomonas paucimobilisATCC 202027 (Previously Identified as Pseudomonas SC 16113)

Fermentation process for growth of Sphingomonas ATCC 202027 wasdeveloped for the production of dipeptide aminotransferase. Dipeptideaminotransferase, an intracellularcellular enzyme used for the oxidationof N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-](phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid,discovered during screening of the Bristol-Myers Squibb culturecollection and environmental isolates. The fermentation process wasscaled-up to 500 L. The highest aminotransferase activity of 231 mg of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid,formed/hr/gram of protein was achieved after 48 hours growth period.Starting from the cell-extracts, the aminotransferase was purified 354fold with a specific activity (mg product formed/hr/g of protein) of36600. The purification procedure included DE-52 ion-exchange columnchromatography, phenyl sepharose column chromatography and sephacrylS-200 column chromatography. The purified enzyme after sephacryl S-200column chromatography showed a single protein band on SDS/PAGE using asilver stain. The molecular weight of enzyme as determined bygelfiltration techniques is 81,000 daltons and subunit size asdetermined SDS/PAGE is 40,000 daltons indicating that the dipeptideaminotransferase is a dimeric protein. The amino terminal and internalsequences of the purified protein were determined for cloningaminotransferase gene.

Growth of Pseudomonas sp. SC 16113 in Shake-flasks

One vial of organism was used to inoculate 100 ml of medium A containing1.5% peptone, 0.01% yeast extract, 0.2% KH₂PO₄, 0.2% K₂HPO₄, 0.01% MgSO₄and 0.2% NaCl. Cultures were grown at 28° C. and 280 RPM for 48 to 72hours on a rotary shaker. One hundred mL of this culture was transferredto one liter of medium A. Cultures were grown in 4-L flask at 28° C. and250 RPM for 48 hours on a rotary shaker. Cultures were harvested bycentrifugation at 18,000×g for 15 minutes, cells were recovered andstored at −70° C. until used.

Preparation of Cell Extract

Preparation of cell extracts were carried out at 4-7° C. Cells werewashed with 25 mM potassium phosphate buffer pH 8.0 (buffer A) andwashed cells (2 g) were suspended in 10 mL of buffer A containing 10 mMNa-EDTA. To the cell suspensions, 0.1 mL of 100 mM PMSF solution inisopropanol and 0.1 mL of 0.5 M DTT were added. Cell-suspensions (20%W/V, wet cells) were passed through a French Press at 15,000 psipressure and disintegrated cells were centrifuged at 25,000×g for 30 minat 4° C. The supernatant solution obtained after centrifugation isreferred to as cell extracts. Cell suspensions of more than 100-mlvolumes were disintegrated with a Microfluidizer (Microfluidics, Inc) at12,000 psi (two passage) and disintegrated cells were centrifuged at25,000×g for 30 min to obtain cell extracts.

Enzyme Assay

The reaction mixture in 5 mL contained 2.7 mL of cell extracts, 30 μL of10 mM pyridoxal phosphate, 1.95 mL of dipeptide dimer solution (50 mg/mLstock solution), and 75 μL of sodium α-ketoglutarate (80 mg/mL stocksolution). The reaction mixture was incubated at 30° C. at 100 RPM. At1, 4 and 16 hours, 0.95 mL of samples were taken and 0.05 mL of TCA(100% w/v) was added. After one hour incubation with TCA at roomtemperature 1 mL of acetonitrile was added to the solution. Samples wereanalyzed for N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide and[[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, concentration by HPLC. Protein concentration in cell extract wasdetermined by Bio-Rad protein reagent using bovine serum albumin as astandard. The assay mixture contained 1-10 μL of enzyme fraction, 0.8 mLwater and 0.2 mL Bio-Rad reagent. After through mixing the absorbance ofsolution was measured at 595 nm. The concentration of protein wascalculated from the standard graph derived with bovine serum albumin asstandard protein.

Growth of Sphinpomonas paucimobilis. ATCC 202027 in a Fermentor

Sphingomonas paucimobilis. ATCC 202027 were grown in 700-L fermentorscontaining 500 L of medium A containing 1.5% peptone, 0.01% yeastextract, 0.2% KH₂PO₄, 0.2% K_(2HPO) ₄, 0.01% MgSO₄, 0.2% NaCl, 0.025%SAG and 0.025% Dow Corning antifoam. Growth consisted of two inoculumdevelopment stages and one fermentation stage. Inoculum developmentconsisted of F1 and F2 stages. In the F1 stage, a frozen vial ofSphingomonas paucimobilis. ATOC 202027 was inoculated into 100 ml ofmedium B containing 1% glucose, 0.1% KH₂PO₄, 0.1% K_(2HPO) ₄, 0.05%MgSO₄, 0.05% yeast extract, 0.05% (NH4)₂SO₄, 0.05% NaCl, and 0.01%CaCl₂. The growth was carried out in 500-ml flasks at 28° C. and 250 RPMfor 24 hours. In the F2 stage, 100-mL of F1 stage culture of organismwas inoculated into 1 L of medium B in a 4-L flask and incubated at 28°C. and 250 RPM for 48 hours. Fermentor containing medium A wasinoculated with 4 L of F2 stage inoculum and grown at 28° C. and 220 RPMagitation with 250 LPM (liter per minute) aeration and 10 PSIG backpressure. During fermentation, cells were periodically harvested bycentrifugation from 200 ml of culture broth. Cell extracts were preparedas described earlier to assay for enzyme activity. The specific activity(mg of product formed/hr/g of protein) was determined.

HPLC Analysis forN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide and[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid

HPLC analysis was performed using a Hewlett-Packard (HP) 1090 with aVydac C-18 reverse phase column. The mobile phase solvent A containing0.1% trifluoroacetic acid (TFA) in water and solvent B containing 0.1%TFA in 70% acetonitrile: 30% water. The following gradient of solvent Aand B was used for the separation ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide and[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid.

  0 min 100% A  0-15 min  50% B 15-25 min 100% B 25-26 min  0% B 26-30min  0% B

The flow rate was 1 mL/min. The column temperature was ambient, and thedetection wavelength was 215 nm. Under this conditions, the retentiontimes for N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide was 17.8 min and for[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid was 20.1 min under above conditions. The retention times formonomer of N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide is 15.3 min.

Purification of Aminotransferase

Preparation of cell extracts were carried out at 4-7° C. Cells werewashed with 25 mM potassium phosphate buffer pH 8.0 (buffer A) andwashed cells (200 g) were suspended in 1L of buffer A containing 10 mMNa-EDTA. To the cell suspensions, 0.1 mL of 100 mM PMSF solution inisopropanol and 0.1 mL of 0.5 M DTT were added. Cell-suspensions (20%W/V, wet cells) were disintegrated with a Microfluidizer (Microfluidics,Inc) at 12,000 psi (two passage) and disintegrated cells werecentrifuged at 25,000×g for 30 min to obtain cell extracts. Thepurification of aminotransferase was carried out by following steps.

(1) DEAE-cellulose Column Chromatography

The cell extract was loaded on to Whatman DE-52 column (400 mL packedbed) eqilibrated with buffer A. The column was washed with 400 mL ofbuffer A and then with 400 mL of buffer A containing 0.2 M NaCl. Enzymeactivity was eluted with a 2L gradient of buffer A containing NaCl from0.2-0.6 M. Fractions of 20 mL were collected. Fractions containing thehighest specific activity were pooled.

(2) Phenyl Sepharose Column Chromatography

The pooled fraction from DE-52 column was adjusted to 1M ammoniumsulfate (132 g/L ammonium sulfate added) and loaded on to Pharmacia fastflow Phenyl Sepharose column (150 mL bed volume) equilbrated with bufferA containing 132 g/L ammonium sulfate (1M ammonium sulfate). The columnwas washed with 150 mL of buffer A containing 1M ammonium sulfate andthen with buffer A containing 0.25 M ammonium sulfate. The enzymeactivity was eluted with a 400 mL gradient of buffer A containingammonium sulfate from 0.25 M-0 M ammonium sulfate. Fractions of 20 mLwere collected. The most active fractions were pooled and concentratedby ultrafiltration to 4 mL using an Amicon YM -10 membrane filtration.

[3] Sephacryl S-200 Column Chromatography

The concentrated fraction (4 mL) from Amicon step was loaded on toSephacryl S-200 column (450 mL) equilibrated with buffer A. Fraction of10 mL were collected.

Sodium Dodecyl Sulfate Polvacrylamide Gel-electrophoresis (SDS/PAGE)

The active fractions from Sephacryl S-200 column were evaluated bySDS-PAGE as described in the PhastSystem procedure by Pharmacia, usingthe homogeneous 12.5% Phastgel. The enzyme samples were added to abuffer containing 10 mM Tris-HCl, 1 mM EDTA (ethylenediamine tetraaceticacid) pH 8, 2.5% SDS and 5% β-mercaptoethanol. The mixture was heated at100° C. for 5 minutes, and bromophenol blue was added to 0.01%. Gelswere stained with silver stain and destained in 10% acetic acidsolution. Marker with standard molecular weight contained phosphorylaseβ(94,000), bovine serum albumin (67,000), ovalbumin (43,000), cabonicanhydrase (30,000), soybean trypsin inhibitor (20,100), α-lactabumin(14,400).

Determination of Molecular Weight of Aminotransferase

The molecular weight of aminotransferase was determined by Pharmaciasize exclusion chromatography using a Superose-12 column (15 cm×1 cm).The column was equilibrated with buffer A. The Aminotransferase(Sephacryl S-200 fraction) was applied to the column and eluted with thebuffer A at a flow rate of 0.4 mL/min. Fractions of 1 mL were collected.Standard protein mixture containing thryglobulin (669,000 MW), ferritin(440,000 MW), Human IgG (150,000 MW), human transferrin (81,000 MW),Ovalbumin (43,000 MW), Myoglobin (17,600 MW), and Vitamin B12 (1355 MW)was also applied to the column and eluted with buffer C. Molecularweight of glutamate oxidase was determined from standard graph.

Aminotransferase activity from cell extracts of Pseudomonas sp. SC 16113grown in shake-flask cultures were 0.2 mg of product [4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]aminol-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, methyl ester formed/hr/mL of extract. All activity was obtained inthe cells after centrifugation of culture broth indicated that theenzyme is intracellular protein.

Aminotransferase was purified from cell extracts of Sphingomonaspaucimobilis ATCC 202027 (Table 1)

The purified protein (CM cellulose fraction 17 & 18) gave a single bandof protein on a SDS/PAGE corresponding to a molecular weight of 40,000daltons. The molecular weight of purified protein was determined bygel-filtration on a Superose-12 column and FPLC system. The molecularweight of purified protein is 81000 daltons indicating that dipeptideaminotransferase is a dimeric protein.

The aminoterminal and internal sequence of purified dipeptideaminotransferase was determined to synthesize oligonucleotide probes forfurther cloning of enzyme.

TABLE 1 Total Total Sp. Activity Volume Protein Activity (Units/Purification Step (mL) (mgs) (Units) mgs) (fold) Cell extract 800 2544400 157 1 DE-52 150 57.3 219 1462 9.3 Phenyl- 120 5.04 93 18476 117sepharose Sephacryl 40 1.32 53 3666 254 S-200

EXAMPLE 4 Production of[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b](1,3]thiazepine-7-carboxylicacid, from N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide using Aminotransferase from Pseudomonas sp. SC16113(Sphingomonas paucimobilis ATCC 202027)

Reaction:

Preparation of Cell-free Extracts

Approximately 700 g of Pseudomonas sp. SC 16113 cells (XP9540; Rec 50;Tank 122) were suspended in 3.5 L 0.01M phophate buffer, pH 8; 5 mM EDTAand homogenized with a Tekmar laboratory homogenizer. These cells werethen centrifuged at 4° C. for 30 min at 12000 rpm. The supernatent wasthen discarded. The cells were resuspended in 3.5 L 0.1M phophatebuffer, pH 8; containing 5 mM DTT, 1 mM PMSF, and 5 mM EDTA. Thehomogenized cell-suspensions were then passed twice throughMicrofluidzer (200 m chamber) at 7500 psi to prepare the cell-freeextracts. The chamber and the cell-suspensions were chilled to 4° C.prior to the cell disruption. The cell extract was then centrifuged toremove the debri (4° C., 13000 rpm, 3 hour).

Reaction Conditions

To the 2 L of cell-free extracts, 10 ml each of 10 mM pyridoxalphosphate, α-ketoglutarate (80 mg/ml) and NAD (20 mg/ml) were added. Thereaction mixture was mixed gently with an overhead teflon agitator (200rpm). To the stirring reaction mixture, 120 ml ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide solution (50 mg/ml) was added. The reaction wascarried out at ambient temperature. Aliquots were taken out every 0.5hrs to measure the amount of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid formed. After 1.75 hours of reaction, 200 ml of TCA solution (100%v/v) was added, and after mixing the pH of the resulting solution was3.1. To the reaction mixture 1.8 L of acetonitrile was added mixedgently for 15 minutes, then let stand at room temperature for 2.30hours. The quenched reaction mixture was then centrifuged to removeprecipitated proteins at 4° C. at 13000 rpm for 1 hr. Theaqueous/organic supernate was used for isolation of product[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid.About 40M % yield of product was obtained.

EXAMPLE 5 Cloning and Expression of Aminotransferase from Sphinglomonaspaucimobilis ATCC 202027 in to Escherichia coli

Amplification of a PCR fragment corresponding to the Sphingomonaspaucimobilis ATCC 202027 aminotransferase gene

The lysine aminotransferase protein (LAT) was found to convertN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]L-lysine,(1->1′)-disulfide to (6). The lysine aminotransferase protein (LAT) waspurified from Sphingomonas paucimobilis ATCC 202027 and its aminoterminal and internal peptide sequences were determined. A mixedoligonucleotide based on the amino terminal sequence was synthesizedtaking into consideration the high G+C content of the Sphingomonaspaucimobilis ATCC 202027 genome.

Ile Thr Pro Leu Met Pro Val Tyr ATC/T ACC/G CCG C/TTG ATG CCG GTC/G TAT

The DNA sequence is SEQ.ID.NO.:3 and the amino acid sequence isSEQ.ID.NO.:4

The downstream (antisense) primer based on a conserved amino acidsequence found in other aminotransferases was(Tyr-Gly-Asn-Pro-Leu-Ala)(SEQ.ID. NO.:5), with a corresponding oligomerof GCG/C AGC/G GGG TTC/G CCC/G CC (antisense) (SEQ.ID.NO.:6). A PCRreaction using Tth polymerase was performed to obtain a 850 base pair(bp) fragment. This fragment was isolated and cloned into plasmid vectorpCRII. The presence of the insert was verified using PCR and restrictiondigests, and the sequence of the insert was determined using anALFexpress automated DNA sequencer. This sequence demonstrated stronghomology to bacterial aminotransferases found in GenBank database. Aninternal peptide sequence of 19 amino acids obtained from a trypticpeptide of purified aminotransferase protein from Sphingomonaspaucimobilis ATCC 202027 was also located. These data indicated that thePCR fragment amplified was in fact representative of part of the bonafide LAT enzyme.

Isolation of the Complete lat Gene

The 850-bp PCR fragment was used as a hybridization probe to identifythe entire LAT gene. Sphingomonas paucimobilis ATCC 202027 chromosomalDNA was purified and partially digested with restriction endonucleaseSau3A1.

Fragments of 6-10 kilobases (kb) were extracted from an agarose gelfollowing electrophoresis and ligated to BamHI-cleaved plasmid vectorpZerol. The DNA was transformed into E. coli TOP 10 F′ cells byelectroporation and selected on LB medium containing the antibioticZeocin. Transformants were then transferred onto nylon filters and lysedin situ. Following hybridization using a ³²P-labeled PCR fragment,several strongly hybridizing colonies were seen. They were picked fromthe master plate and grown in liquid medium for plasmid DNA isolation.To confirm that these plasmids did in fact contain the lat gene, twocriteria were used: (1) The DNA must possess a 669-bp internal Ncolfragment which is found in the PCR fragment and (2) the DNA must serveas a template for amplification using primers specific to the 850-bp PCRfragment. One colony contained a plasmid which met both these criteria.It contained an insert of approximately 6.3 kb and was thus namedpLAT6.3.

Sequencing of the Complete lat Gene

The entire aminotransferase gene of pLAT6.3 was sequenced (FIG. 1). Atypical gram-negative promoter and ribosome binding site followed 9bases later by an initiation codon ATG (Methionine) was found. The sizeof the coding region of the gene is 1221 bp. Based on our computeranalysis of the gene, this region should encode 398 amino acids with amolecular weight of 42,457 daltons. We identified additional runs ofamino acids which matched that obtained from internal peptide sequencingof the purified LAT protein. This information confirmed that the entirelat gene was present on pLAT6.3 and that it encoded the same proteinisolated from Sphingomonas paucimobilis ATCC 202027.

Cloning and Overexpression of the lat Gene in E. coli

The polymerase chain reaction was used to precisely amplify the lat genewhich also contained restriction sites at both ends for cloning intoexpression plasmids. For digestion and ligation into pKK223-3, the latgene was amplified with EcoRI and BamHI sites at the 5′ and 3′ ends.Similarly, Ndel and Xbal sites were added to the 5′ and 3′ endsrespectively for ligation into pAL781. Both the amplified fragment andthe vector DNAs were cleaved with the appropriate enzymes and ligatedtogether. The plasmids formed were named pkk 223-3-LAT and pAL781-LAT/Arespectively. The ligation samples were electroporated into E. colistrains TOP 10 F′ (pKK223-3-LAT) or GI724 (pAL781-LAT/A). The presenceof the lat gene in the recombinant plasmids was confirmed using PCR withlat-specific primer and restriction digestion analysis.

TOP 10 F′(pKK223-3-LAT) was grown in LB medium (tryptone, 1.0%; yeastextract, 0.5%; NaCl, 1.0%) containing 100 Tg/ml of ampicillin. At anOD600 of ca. 1.0, the tac promoter controlling expression was inducedwith 100 TM IPTG. Samples were taken 1, 2, and 3 hr post-induction andanalyzed for aminotransferase activity and used in the bioconversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid,(table 2).

GI724(pAL781-LAT/A) was grown for 18-20 hr in MRM (0.6% Na₂HPO₄, 0.3%KH₂PO₄, 0.125% (NH₄)₂SO₄, 2% Casamino acids [Bacto grade], 1.0%glycerol, 1 mM MgSO4). The culture was then inoculated into MIM mediumat a starting OD₆₀₀ of 0.20 (MIM contained 0.6% Na₂HPO₄, 0.3% KH₂PO₄,0.125% (NH₄)₂SO₄, 0.2% Casamino acids [Bacto grade], 0.5% glucose, and 1mM MgSO4). Ampicillin was added to all media at a final concentration of100 Tg/ml. At an OD₆₀₀ of ca. 0.5, L-tryptophan from a filter-sterilized10 mg/ml solution was added to a final concentration of 100 Tg/ml.Samples were removed at 3, 6, and 22 hr post-induction foraminotransferase activity and used in the bioconversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid.

Both recombinant strains were able to convertN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid (Table 2), while control culture which did not possess the lat geneshowed no such activity. Expression using the tryptophan-induciblepromoter was better than that using the tac promoter so the formerstrain was investigated in more detail. Activity was seen at all timepoints but peaked at 6 hr post-induction (Table 2). Coincidentally,extracts prepared from the induced culture revealed a strong proteinband of the same molecular weight as the purified aminotransferase at 6hr post-induction. Further experiments indicated that most of thisprotein was in the intracellular soluble fraction.

A kanamycin-resistant version of the above plasmid was also created.Plasmid pET9b was excised by digestion with restriction endonucleasesAlwNI and EcoRI. A 1171-bp fragment containing the Kanamycin resistant(Km^(R)) gene was purified and the ends made blunt-ended by treatmentwith Klenow DNA polymerase plus all four deoxyribonucleotides. Themodified fragment was ligated into pAL781-LAT/A which had been digestedwith Sspl. After electroporation into GI724, kanamycin-resistantcolonies were picked and verified for the presence of both the Kn^(R)and lat genes. This plasmid was named pAL781-LAT/AK. A fermentationidentical to the one performed for the ampicillin-resistant version ofthe plasmid was carried out. Assay results as shown in the Table 2.

TABLE 2 Culture Wet Cells Growth Compound B Weight Sample (hours) formed(mg/mL) (mgs) TOP 10 F′(pKK223-3-LAT) 3 0.126 157 G1724(pAL781-LAT/A) 60.422 250 G1724(pAL781-LAT/A) 2 0.195 420 4 0.266 340 6 0.308 300G1724(pAL78I-LAT/AK) 2 0.193 330 4 0.311 300 6 0.292 390 Contol (withoutlat gene) 6 0 70 **Compound B is [4S-(4I, 7I,10aJ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid,

EXAMPLE 6 Production of Aminotransferase (Cloned from Sphingomonaspaucimobilis ATCC 202027 in to Escherichia coli GI724 (pAL781-LAT)

The process described is for the growth of E. coli GI724(pAL781-LAT) in20 liter fermentors. The E. coli cells produce an aminotransferaseenzyme which is used for the enzymic conversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid.

The aminotransferase enzyme is produced intracellularly. The recoveredcell paste can be used to prepare a cell-free extract for thebiotransformation.

Process Details for Growth of Escherichia coli

E. coli cultures were first grown in a 4-L flask containing 1-L mediumfor 24 hours and 24-hours grown cultures were inoculated into 25-Lfermentor containing 16 L medium. Medium composition and growthconditions are as follows:

The strain approved for all fermentations is E. coliGI724(pAL781-LAT/A). A kanamycin-resistant derivative of pAL781-LAT thatalso overexpresses the enzyme in E. coli GI724 is available and is beingtested at the 16-liter scale.

Stage One—F1 Stage

1. Two 1 ml vials are inoculated into each 4 liter flask (two are neededfor a 16 liter fermentor) containing 1 liter of MT3 medium. Each flaskis incubated at 30° C. for 24 hours at 250 RPM.

MT3 medium: 1.0% NZ Amine A 2.0% Yeastamine 2.0% Glycerol 0.6% SodiumPhosphate dibasic 0.3% Potassium Phosphate monobasic 0.125% Ammoniumsulfate 0.0246% Magnesium sulfate heptahydrate 0.01% Ampicillin, sodiumsalt Batched with deionized water and autoclaved for 20 minutes at 121°C.

Stage Two—Fermentation Stage

Medium in the fermentor is batched with deionized water to a finalvolume of 15 liters with the following ingredients:

1.0% NZ Amine A 2.0% Yeastamin 2.0% Glycerol 0.6% Sodium Phosphatedibasic 0.3% Potassium Phosphate monobasic 0.125% Ammonium sulfate 0.05%Polypropylene glycol

The pH after batching is adjusted to 7.0-7.2. Sterilization is conductedat 121.5° C. for 20 minutes by jacket steam only. After sterilization,the temperature is reduced to 37° C. and the volume is approximately 16liters. Prior to inoculation, a solution containing magnesium sulfateand ampicillin are added aseptically (after filter sterilization) to thetank to a final concentration of 1 mM and 100 mg/L, respectively. Thefermentor is inoculated with broth from 1 or more F1 flasks to yield anequivalent optical density in the tank of 0.25 at Log 0. Approximately,1.5 liters of inoculum (1½ F1 flasks) will be needed for a 16 literworking volume fermentor. Note, all filter sterilization of mediumcomponents is done with a 0.2 micron cellulose nitrate filter. Opticaldensity is measured with a spectrophotometer.

Fermentor Operating Parameters

16 liter working volume

Temperature: 37° C.

Aeration: 0.5 volume air per working volume per minute

Pressure: 690 mBar

Agitation: 250 RPM

There is no pH control and foam is controlled by addition of UCON ondemand.

Samples are taken during the course of the fermentation for pH, partialvolume of solids, relative viscosity, optical density, wet cell weightsand enzyme activity. The optical density of properly diluted (with log Mmedium) samples is measured at 600 nm on a spectrophotometer which hasbeen blanked with log M medium. Off gas CO₂ from the fermentor ismonitored continuously with a gas analyzer. The fermentor runs for 6-8hours. At that time the tank is subcooled to 10° C. while maintainingthe same agitation and aeration (in overlay) and then recovered bycentrifugation.

Yields and Purities

Four batches of E. coli have been run based on this PreliminaryLaboratory Process Description. The results of these batches aresummarized below.

Batch No. XP9682 XP9683 XP9683 XP9683 Tank No. BF#3 BF#3 BF#4 BF#5Fermentor Volume (L) 20 20 20 20 Working Volume (L) 16 16 16 16 HarvestLog (hr) 6 6 8 6 Aminotransferase 1.01 1.01 1.67 1.67 Activity [90 min.assay] [70 min. (mg product/g wet assay] cell/hr) Harvested Wet Cell 9.18.9 8.8 9.4 Weight (g/L)

Preparation of Cell-free Extracts

15 g of centrifuged wet cell paste were suspended in 100 ml of 100 mMphosphate buffer pH 8.0, 10 mM Na EDTA and homogenous cell suspensionswere using a homogenizer. To the cell suspensions 0.1 ml of 0.1 M PMSFsolution in isopropanol and 0.1 ml of 0.5 M DTT solution were added.Cell suspensions were passed through a French Press at 15,000 PSIpressure and disintegrated cells were centrifuged at 25,000×g at 4° C.The supernatant solution obtained after centrifugation is referred to ascell extracts. Cell suspensions of more than 100 mL volumes weredisintegrated with a Microfluidizer at 12, 000 PSI pressure (twopassage) and disintegrated cells were centrifuged at 25,000×g for 30 minto obtain cell extracts.

Enzyme Assay

The reaction mixture in 1 mL contained 0.1 mL of cell-free extracts, 0.8ml of 100 mM phosphate buffer pH 8.0 containing 10 mM Na EDTA, 10 μl of0.5 M DTT solution, 10 μl of 10 mM PLP solution, 25 μl ofα-ketoglutarate solution and 0.65 μl of 50 mg/mL of dipeptide dimersolution. Reaction mixtures were prepared in tubes. Tubes were incubatedat room temperature on a end-over-end shaker. Samples were taken at 0.5,1.5 and 6 hours. Quench the enzyme reaction mixture by adding 0.2 ml oftrichloroacetic acid and 1.8 ml of acetonitrile to the reactionmixtures. Let mixture stand at room temperature for 2-4 hours andanalyzed by HPLC for substrate and product concentration as follows:

Analytical Methods

Filter the quenched reaction mixture to a HPLC vial, using a Lid/X typesyringe filter. The concentration of substrate and product aredetermined by the following HPLC method.

Column Vydac C-18 Buffers A: 0.1% TFA in H₂O B: 0.1% TFA in 70%Acetonitrile, 30% H₂O Gradient  0-15 min: 50% B 15-25 min: 100% B 25-26min: 0% B 26-30 min: 0% B Flow Rate 1 ml/min. Detector UV-215 nm. Thedimer(N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide)elutes at 15.3 min and the product (6) elutes at 20.1 min under theseconditions.

Determination of Protein Content of the Cell-free Extracts

Bio-Rad protein assay was used for determining the protein quatitation.The assay was carried out as described by the manufacturer (Bio-Rad).The assay consists of adding 5 μL of cell-free extract in a total volumeof 0.8 mL water. The Bio-Rad reagent (0.2 mL) is added to the 0.8 mL andafter thorough mixing the absorbance of the solution was measured at 595nm. The concentration of the protein was read from the standard curvewith bovine serum albumin (BSA) as the standard protein.

Unit of activity=μmoles of product formed/min/per mL of cell extracts.

EXAMPLE 7 Preparation of[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid, fromN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide

Introduction

The process described is the conversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, by oxidative deamination using cloned transaminase fromSphingomonas paucimobilis (previously identified as Pseudomonas sp) SC16113 expressed in E. coli GI724[paI781-LAT]SC16240. The reactionrequires α-ketoglutarate, as the amine acceptor. DTT is used to reducethe dimer N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to monomer. Glutamate produced during the reaction isrecycled to α-ketoglutarate by the oxidative deamination using glutamateoxidase from Streptomyces sp. obtained from Sigma Chemicals, St. Louis,Mo. (Product #G0400). The expected reaction molar yield is 70-80 M %.The procedure described uses cell-free extracts for transaminase, andglutamate oxidase.

Raw Materials for 22.2 g Batch: Material Source Mol. Wt. Amount (4)38449-156-29 794.11 22.2 g α-Ketoglutarate Sigma 63.06 19.5 g Pyridoxalphosphate Sigma 247.1 100 mg Dithiothreitol Sigma 154.2 7.03 g K₂HPO₄Fisher 174.2 16.1 g KH₂PO₄ Fisher 136.14 16.1 g EDTA Sigma 292.2 3.65 gPMSF Sigma 174.2 87 mg Trichloroacetic acid Sigma 114 600 mL (TCA) NaOHFisher 40 Acetonitrile VWR 41.05 5 L Transaminase from 200 Units E. coliGI 724 Cell extract [pAL781-LAT] (SC16240) Glutamate oxidase 8150 Unitsfrom Streptomyces sp. (partially purified) (Sigma Chemicals) Water,deionized 4.5 L

Process Details

The process details for the reaction steps based on 22 gN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide input.

(1) Transaminase

Preparation of Cell-free Extracts

75 g of wet recombinant E. coli cells GI 724[pAL781-LAT] (SC16240)(XP9676; Rec126) were suspended in 500 mL of 0.1 M phosphate buffer, pH7.8 containing 5 mM DTT, 1 mM PMSF, and 5 mM EDTA in a 1 L beaker.Cell-suspensions were homogenized with a Tekmar laboratory homogenizer.Cell-suspensions were passed twice through Microfluidzer at 7500 psi and4° C. Disrupted cell-suspensions were centrifuged at 4° C. at 13000 rpmfor 25 min. to remove the debris.

(2) Reaction Conditions

22.2 g of N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide into a 5 L bioreactor. 2.5 L of phosphate buffer pH7.8 containing 10 mM EDTA to the bioreactor. The reaction mixture wasagitated at 200 rpm. Gradually increase the pH of the above solution to12.0 by slow addition of 5N NaOH, while stirring the solution at roomtemperature. Maintained the pH at 12.0 with the addition of 5N NaOH,until all solid was dissolved. The pH was readjusted to 7.8 with slowaddition of conc. H₃PO₄. The temperature of the reaction mixture wasmaintained at 30° C. with a water bath. To the reaction mixture, 6.75 gof DTT, 19.5 g of α-ketoglutarate, disodium salt and 100 mg of pyridoxalphosphate were added. Additional 1.4 L of 100 mM phosphate buffer, pH7.8 containing 10 mM EDTA was added to the reaction mixture. Thereaction was started by the addition of 100 mL of cell-extracts oftransaminase [aminotransferase (1 unit/mL activity) from Escherichiacoli]. 300 mL of Glutamate oxidase solution (7 U/mL) was added at 30minutes, 1 hr, 1.5 hrs, and 3.5 hrs. The pH was maintained at 7.8 withthe addition of 5N NaOH. Periodically samples of 1 mL were removed andquenched with 0.1 mL of trichloroacetic acid and 0.9 mL of acetonitrile.The sample was allowed to stand at room temperature for 2 h, thenfiltered and analyzed by HPLC to measure the amount of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, formed. After 6 hours of reaction time, 0.6 L of TCA solution(100% v/v) was added to the reactor to terminate the enzymatic reaction.The solution was mixed for 15 minutes, and let stand at room temperaturefor 3 hrs. To the reaction, 5L of acetonitrile was added and the amountof product formed was then obtained by an HPLC quantiation of analiquot.

Yields Input Compound A* Compound B** Yield Experiment # (g) (g) (g) (M%) 40455-23  0.30 0.07 0.19 64.2 40455-24  3.00 0.83 1.90 66.5 40455-41 5.00 1.35 2.92 65.0 40455-43 12.20 4.30 8.00 71.0 40437-28-15 22.504.70 15.5 70.0 *Compound A isN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine, (1->1′)-disulfide **Compound B is [4S-(4α, 7β,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid.

Enzymatic conversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid was carried out using transaminase from rE. coli. Theamino-acceptor α-ketoglutarate, was regenerated using partially purifiedglutamate oxidase from Streptomyces sp. obtained from Sigma ChemicalsCo., St. Louis, Mo. (Product #G0400). The reaction was carried out with3.0 g and 5.0 g ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide in 1 L of 100 mM phosphate buffer. Glutamate oxidase(570 units) was added periodically at 30, 60, and 210 min. After 300 minof reaction time, >66 M % yield was observed. The mass-balance was >95%.

In small scale reactions, (0.1 L) the input ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide was increased to 5 g/L. Using standard bioconversionconditions, the yield of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, was observed to be >70%.

The preparative scale enzymatic conversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide (5 g/L) to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, was conducted in 2.5 L volume using transaminase from rE. coli andglutamate oxidase from Sigma Chemicals Co. The input ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide was 12.2 g. Using standard bioconversion conditions,7.0 g of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, was prepared. The yield was 11 M %.

A second preparative scale enzymatic conversion ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide (5 g/L) to[[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylic acid, wasconducted in 4 L volume using transaminase from rE. coli and glutamateoxidase from Sigma Chemicals Co. The input ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide was 22.2 g. Using standard bioconversion conditions,14.4 g of [4S-(4α, 7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, was prepared. The yield was 67 M %.

EXAMPLE 7a Preparation of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,-1-b][1,3]thiazepine-7-carboxylic acid,from N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide

Reaction in the Absence of Glutamate Oxidase

The process described is the conversion ofN²-[N-[(Phenylmethoxy)carbonyl]]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-](phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiapezine-7-carboxylicacid by oxidative deamination using cloned transaminase fromSphingomonas paucimobilis (previously identified as Pseudomonas sp)SC16113 expressed in E. coli GI724[pAL781-LAT]SC16240. The reactionrequires α-ketoglutarate, as the amine acceptor. DTT is used to reducethe dimer N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to monomer.

Raw Materials for 22.2 Batch: Material Source Mol. Wt. Amount (4)38449-156-29 794.11 22.2 g α-Ketoglutarate Sigma 63.06 19.5 g Pyridoxalphosphate Sigma 247.1 100 mg Dithiothreitol Sigma 154.2 7.03 g K₂HPO₄Fisher 174.2 16.1 g KH₂PO₄ Fisher 136.14 16.1 g EDTA Sigma 292.2 3.65 gPMSF Sigma 174.2 87 mg Trichloroacetic acid Sigma 114 600 mL (TCA) NaOHFisher 40 Acetonitrile VWR 41.05 5 L Transaminase from 200 Units E. coliGI 724 Cell extract [pAL781-LAT] (SC16240) Water, deionized 4.5 L

Process Detail

22.2 g of N²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide into a 5 L bioreactor, 2.5 L of phosphate buffer pH7.8 containing 10 mM EDTA to the bioreactor. The reaction misture wasagitated at 200 rpm. Gradually increase the pH of the above solution to12.0 by slow addition of 5N NaOH, while stirring the solution at roomtemperature. Maintained the pH at 12.0 with the addition of 5N NaOH,until all solid was dissolved. The pH was readjusted to 7.8 with slowaddition of conc. H₃PO₄. The temperature of the reaction mixture wasmaintained at 30° C. with a water bath. To the reaction mixture, 6.75 gof DTT, 195 g of α-ketoglutarate, disodium salt and 100 mg of pyridoxalphosphate were added. Additional 1.4 L of 100 mM phosphate buffer, pH7.8 containing 10 mM EDTA was added to the reaction mixture. Thereaction was started by the addition of 100 mL of cell-extracts oftransaminase [aminotransferase (1 unit/mL activity) from Escherichiacoli]. The pH was maintained at 7.8 with the addition of 5N NaOH.Periodically samples of 1 mL were removed and quenched with 0.1 mL oftrichloroacetic acid and 0.9 mL of acetonitrile. The sample was allowedto stand at room temperature for 2 h, then filtered and analyzed by HPLCto measure the amount of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-phrido-[2,1-b][1,3]thiazepine-7-carboxylic acid,methyl ester formed. After 6 hours of reaction time, 0.6 L of TCAsolution (100% v/v) was added to the reactor to terminate the enzymaticreaction. The solution was mixed for 15 minutes, and let stand at roomtemperature for 3 hours. To the reaction, 5 L of acetonitrile was addedand the amount of product formed was then obtained by an HPLCquantiation of an aliquot.

Yields Compound B Compound A. Input Compound A Compound B YieldSubstrate # (g) (g) (g) (M %) 22.2 4.9 17.2 73

Evaluation of Different Compounds as Substrate for Transaminase

Two new dipeptides N-[N-[(Phenylmethoxy)carbonyl]-L-methionyl]-L-lysine(9) andN<2-[S-Acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (10)were evaluated as substrates for dipeptide aminotransferase by cell-freeextracts of Spongomonas paucimobilis ATCC 202027 in the presence ofα-ketoglutarate. Glutamate oxidase was not used. As no product markerswere available, the formation of new compounds from the enzymaticreaction were investigated by LC-MS. In case of (9) the data indicatesthe formation of a new compound with mol wt. of 392, the compound wasassigned the tentative structure 1. The ε-NH₂ group of (9) was oxidizedand in the presence of TCA the aldehyde cyclized to the enamide withloss of water.

When (10) was treated with cell-free extracts and α-keto-glutarate,several new components were observed by LC-MS. The component with molwt. 420.5 was assigned structure 2, formed by the oxidation of the ε-NH₂group of (3)N<2-[S-Acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine andsubsequent dehydration to produce the cyclic enamide, the component withmol wt. 397 was proposed as Des-acetyl (3)N<2-[S-Acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine. TheDes-acetyl (3)N<2-[S-Acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine andwas then oxidized by the enzyme to[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, mol wt. of 378, as shown in the scheme below.

(3) Evaluation of N-Phenylacetyl Analog (4) as a Substrate forTransaminase

Reaction Conditions

The substrate 5, (conc. 5 mg/mL), namely the phenylacetyl analog, wasincubated with recombinant transaminase in phosphate buffer, pH 8containing 5 mM DTT, 1 mM Pyridoxal phosphate. The reaction was carriedout in the absence of glutamate oxidase with 40 mg/mL α-ketoglutarate at38° C. After 4H, the reaction was terminated with 10% TCA and 90%Acetonitrile. The product was identified as (6)[4S-4R*,7R*,10aR*)]-2,3,4,5,8,9,10,10a-Octahydro-4-[(phenylacetyl)amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, with a yield of 70%.

(4) Evaluation of N-Phenoxyacetyl Analog (7) as a Substrate forTransaminase

The substrate,N²-[N-[(Phenoxyacetyl)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide, compound 7, which is an analog ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide (Phenoxyacetyl was used in place of Cbz) was incubatedwith recombinant transaminase in the presence of DTT and α-ketoglutarate(the amino acceptor). After 6 hours of incubation, a new peak at 17.03min. was observed. The new peak was identified as the product 8,[4S-(4R*,7R*,10aR*)]-2,3,4,5,8,9,10,10a-Octahydro-4-[(phenylacetyl)amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid and based on the mass spec analysis and later conformed with anauthentic standard.

Single Enzyme Process

To reduce the cost of producing two enzymes, the transaminationreactions were carried out in the absence of glutamate oxidase andhigher levels of α-ketogultarate. Historically, the reaction yield inthe absence of glutamate oxidase averaged 33 M %. At 40 mg/mL ofα-keto-glutarate (a 10× increase in concentration in the absence ofglutamate oxidase in reaction mixture), at 40° C., the reaction yieldincreased to 70 M %.

Replacement of DTT

We have discovered that DTT can be used to cleave the disulfide bond ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide to produce the dipeptide monomer, which is thesubstrate for the tranaminase. It was observed, that tributylphosphinewas as effective as DTT for the dipeptide dimer to monomer conversion.In the presence of 10 mM tri-n-butylphosphine, 3.5 mg/mL ofN²-[N-[(Phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine,(1->1′)-disulfide, 40 mg/mL α-ketoglutarate and 0.1 units oftransaminase, 69 M %[[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid, was obtained.

3. Reduction of TCA

To terminate the transaminase reaction, 10% v/v TCA was used. After someoptimization studies, the amount of TCA required to terminate thereaction was reduced to 5% v/v without loss in yields of[4S-(4α,7α,10aβ)]1-Octahydro-5-oxo-4-[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b][1,3]thiazepine-7-carboxylicacid. It was also observed that methane sulfonic acid or sulfuric acid,or hydrochloric acid or acetic acid or phosphoric acid can be used inplace of TCA.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 1191<212> TYPE: DNA <213> ORGANISM: Spingomonas paucimobilis<400> SEQUENCE: 1atgtcgatta cgccgctcat gcccgtttac ccccggtgcg atgtgcgtcc gg#tccgaggc     60gagggctgct acctgatcgg ggagcgcggc gagcgctatc tcgacttcgc ca#gcggtatc    120gccgtcaatc tgctgggcca tggccacccc aagctggtga agaccattgc cg#atcaggct    180gcgacgctga tgcatatctc caacctctac ggctcgccgc tgggggagga at#ttgcgcag    240aagctggtcg ataacagttt cgcggacacc gttttcttca ccaattcggg tg#ccgaagcg    300gtcgagtgcg cgatcaagac cgcgcgccgc tatcattatg ccaatgggca ag#cgcaccgg    360cacaagatca tcagcttcga caacgccttc cacggccgca cgctgggcac ca#tttcggcg    420accagccagc ccaagatgcg cgacgggttc gagccgctgc tgcccggttt cc#aggtcgtg    480cccttcaacg atctcgacgc ggcgctggcc gcgatcgacg acaataccgc cg#gtttcctg    540ctggaaccgg tgcagggtga aggcggcgtg accccggcaa cccaggcatt cc#tggccggc    600ctgcgcaagg cgtgcgacga gcagggcctg ctgctgatcc tggacgaggt gc#agtgcggc    660tatgcccgta ccggcacctt cttcgcccat gaacaatatg gcgtgacgcc gg#acatcatg    720gcggtggcca agggcatcgg cgcgggcttc ccgctcggcg cctgcctcgc ta#ccgaggat    780gcggccaagg gcatggtgtt cggcacccat ggttccacct atggcggcaa cc#cgctcgcc    840atggcggtgg gcatcgcggt gctggaagag gtgctggcgg acgggttcct gg#agcaggta    900acgcatgggt gccgtctgcg ctctgcgctg gagcagatga tcccgaacca tg#acgacatg    960ttcgaggatg tgcgcggcat ggggctgatg ctgggcgtca agatgaagga cg#cctatgac   1020gcgcgtgcct ttgtcggcca tctgcgcgac caacatgggt tcctgtcggt at#cggcgggc   1080cagaatgtgc tgcgcatcct gccgccgctc gtcatcgacg aaagccatat cg#ccgagtgc   1140atcgagaaga tttccgccgg cgcgcggagc ttcgcggacg ccaaggcggc c #           1191 <210> SEQ ID NO 2 <211> LENGTH: 397 <212> TYPE: PRT<213> ORGANISM: Spingomonas paucimobilis <400> SEQUENCE: 2Met Ser Ile Thr Pro Leu Met Pro Val Tyr Pr #o Arg Cys Asp Val Arg1               5    #                10   #                15Pro Val Arg Gly Glu Gly Cys Tyr Leu Ile Gl #y Glu Arg Gly Glu Arg            20       #            25       #            30Tyr Leu Asp Phe Ala Ser Gly Ile Ala Val As #n Leu Leu Gly His Gly        35           #        40           #        45His Pro Lys Leu Val Lys Thr Ile Ala Asp Gl #n Ala Ala Thr Leu Met    50               #    55               #    60His Ile Ser Asn Leu Tyr Gly Ser Pro Leu Gl #y Glu Glu Phe Ala Gln65                   #70                   #75                   #80Lys Leu Val Asp Asn Ser Phe Ala Asp Thr Va #l Phe Phe Thr Asn Ser                85   #                90   #                95Gly Ala Glu Ala Val Glu Cys Ala Ile Lys Th #r Ala Arg Arg Tyr His            100       #           105       #           110Tyr Ala Asn Gly Gln Ala His Arg His Lys Il #e Ile Ser Phe Asp Asn        115           #       120           #       125Ala Phe His Gly Arg Thr Leu Gly Thr Ile Se #r Ala Thr Ser Gln Pro    130               #   135               #   140Lys Met Arg Asp Gly Phe Glu Pro Leu Leu Pr #o Gly Phe Gln Val Val145                 1 #50                 1 #55                 1 #60Pro Phe Asn Asp Leu Asp Ala Ala Leu Ala Al #a Ile Asp Asp Asn Thr                165   #               170   #               175Ala Gly Phe Leu Leu Glu Pro Val Gln Gly Gl #u Gly Gly Val Thr Pro            180       #           185       #           190Ala Thr Gln Ala Phe Leu Ala Gly Leu Arg Ly #s Ala Cys Asp Glu Gln        195           #       200           #       205Gly Leu Leu Leu Ile Leu Asp Glu Val Gln Cy #s Gly Tyr Ala Arg Thr    210               #   215               #   220Gly Thr Phe Phe Ala His Glu Gln Tyr Gly Va #l Thr Pro Asp Ile Met225                 2 #30                 2 #35                 2 #40Ala Val Ala Lys Gly Ile Gly Ala Gly Phe Pr #o Leu Gly Ala Cys Leu                245   #               250   #               255Ala Thr Glu Asp Ala Ala Lys Gly Met Val Ph #e Gly Thr His Gly Ser            260       #           265       #           270Thr Tyr Gly Gly Asn Pro Leu Ala Met Ala Va #l Gly Ile Ala Val Leu        275           #       280           #       285Glu Glu Val Leu Ala Asp Gly Phe Leu Glu Gl #n Val Thr His Gly Cys    290               #   295               #   300Arg Leu Arg Ser Ala Leu Glu Gln Met Ile Pr #o Asn His Asp Asp Met305                 3 #10                 3 #15                 3 #20Phe Glu Asp Val Arg Gly Met Gly Leu Met Le #u Gly Val Lys Met Lys                325   #               330   #               335Asp Ala Tyr Asp Ala Arg Ala Phe Val Gly Hi #s Leu Arg Asp Gln His            340       #           345       #           350Gly Phe Leu Ser Val Ser Ala Gly Gln Asn Va #l Leu Arg Ile Leu Pro        355           #       360           #       365Pro Leu Val Ile Asp Glu Ser His Ile Ala Gl #u Cys Ile Glu Lys Ile    370               #   375               #   380Ser Ala Gly Ala Arg Ser Phe Ala Asp Ala Ly #s Ala Ala385                 3 #90                 3 #95 <210> SEQ ID NO 3<211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Spingomonas paucimobilis <400> SEQUENCE: 3atyacsccgy tgatgccggt stat           #                  #                24 <210> SEQ ID NO 4 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: Spingomonas paucimobilis <400> SEQUENCE: 4Ile Thr Pro Leu Met Pro Val Tyr 1               5 <210> SEQ ID NO 5<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Spingomonas paucimobilis<400> SEQUENCE: 5 Tyr Gly Asn Pro Leu Ala 1               5<210> SEQ ID NO 6 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Conserved amino acid sequ #ence found in other      amino transferases <400> SEQUENCE: 6gcsagsgggt tsccscc              #                   #                  #   17

What is claimed is:
 1. An isolated DNA sequence encoding theaminotransferase of SEQ ID NO: 2, or the complement thereof.
 2. Anisolated DNA sequence encoding an amino transferase having at least 80%identity to SEQ ID NO: 2, or the complement thereof.
 3. An isolated DNAsequence having the sequence of SEQ ID NO: 1 or its complement.
 4. Anisolated DNA sequence capable of hybridizing under stringent conditionsto a sequence of claim 3, wherein said isolated DNA sequence encodes anaminotransferase or a fragment of an aminotransferase that iscatalytically active, and wherein said stringent conditions are:incubation in 4×SET buffer, 10×Denhardt's solution, 0.1% SDS, 0.1%sodium pyrophosphate, 50 μg/ml denatured salmon sperm DNA at 68° C. for16-24 hours; followed by washing three times for 20 minutes in 3×SET,0.1% SDS, 0.1% sodium pyrophosphate at 68° C., washing twice for 20minutes in 1×SET, 0.1% SDS, 0.1% sodium pyrophosphate at 68° C., washingonce for 20 minutes in 0.1×SET, 0.1% SDS, 0.1% sodium pyrophosphate at68° C., and washing once for 20 minutes in 4×SET at room temperature. 5.An isolated DNA sequence selected from the group consisting of: (a) anisolated DNA sequence encoding a fragment of the amino acid sequence ofSEQ ID NO: 2 that is catalytically active, or the complement thereof;and (b) an isolated DNA sequence capable of hybridizing under stringentconditions to the sequence encoding a fragment of the amino acidsequence of SEQ ID NO: 2 that is catalytically active, or the complementthereof, wherein said isolated DNA sequence capable of hybridizingencodes an aminotransferase or a fragment of an aminotransferase that iscatalytically active, and wherein said stringent conditions are:incubation in 4×SET buffer, 10×Denherdt's solution, 0.1% SDS, 0.1%sodium pyrophosphate, 50 μg/ml denatured salmon sperm DNA at 68° C. for16-24 hours; followed by washing three times for 20 minutes in 3×SET,0.1% SDS, 0.1% sodium pyrophosphate at 68° C., washing twice for 20minutes in 1×SET, 0.1% SDS, 0.1% sodium pyrophosphate at 68° C., washingonce for 20 minutes in 0.1×SET, 0.1% SDS, 0.1% sodium pyrophosphate at68° C., and washing once for 20 minutes in 4×SET at room temperature. 6.An isolated DNA sequence consisting of the sequence of SEQ ID NO:3 orits complement.
 7. A vector comprising the nucleic acid molecule ofclaim
 1. 8. A vector comprising the nucleic acid molecule of claim
 3. 9.A vector comprising the nucleic acid molecule of claim
 4. 10. A vectorcomprising the nucleic acid molecule of claim
 5. 11. A vector comprisingthe nucleic acid molecule of claim
 6. 12. The vector of claim 7, whereinthe nucleic acid molecule is operatively linked to one or moreregulatory DNA sequences.
 13. The vector of claim 8, wherein the nucleicacid molecule is operatively linked to one or more regulatory DNAsequences.
 14. The vector of claim 9, wherein the nucleic acid moleculeis operatively linked to one or more regulatory DNA sequences.
 15. Amethod of producing a host cell comprising genetically engineering cellswith the vector of claim
 7. 16. A method of producing a host cellcomprising genetically engineering cells with the vector of claim
 8. 17.A method of producing a host cell comprising genetically engineeringcells with the vector of claim
 9. 18. A host cell comprising the vectorof claim
 7. 19. A host cell comprising the vector of claim
 8. 20. A hostcell comprising the vector of claim
 9. 21. The host cell of claim 18wherein said host cell is prokaryotic or eukaryotic.
 22. The host cellof claim 19 wherein said host cell is prokaryotic or eukaryotic.
 23. Thehost cell of claim 20 wherein said host cell is prokaryotic oreukaryotic.
 24. The host cell of claim 21 which is E. coli.
 25. The hostcell of claim 22 which is E. coli.
 26. The host cell of claim 23 whereinsaid host cell is prokaryotic or eukaryotic.
 27. A method of producingan aminotransferase comprising culturing a host cell of claim 18 underconditions permitting expression of said aminotransferase.
 28. A methodof producing an aminotranferase comprising culturing a host cell ofclaim 19 under conditions permitting expression of saidaminotransferase.
 29. A method of producing an aminotransferasecomprising culturing a host cell of claim 20 under conditions permittingexpression of said aminotransferase.